Technologies suitable for the preparation of glycoconjugates such as glycopeptides, glycoproteins, glycolipids, glycosylated cell surfaces, glycosylated cell membranes and other glycosylated non-biological surfaces have a great importance in drug discovery and glycobiology. Such enabling technologies play essential roles in the development of glycopharmaceuticals by conjugating immunogenic and non-immunogenic carbohydrate moieties to chemical and/or biological entities. The most advanced technologies are usually based upon the use of ligation chemistries, in which protecting group assistance is avoided during the conjugation of ligating probes to target chemical and biological entities.
Several ligation methodologies have been described in scientific literature focusing on the substitution of N-terminal and lysine side-chains of peptides and proteins in order to deliver the desired carbohydrates moieties. It is well-known that primary amino functional groups are abundant in all kind of biological samples such as organs, skin, fur, silk, cell surfaces and could also be easily displayed on synthetic polymers. Thus, primary amine selective ligation methodologies have the greatest potentials to provide products of many kinds for numerous industries.
Primary amine ligation chemistries have to provide proper reactivities, chemoselectivities, often satisfactory site-selectivities in water or other aqueous solutions while eliminating the occurrence of severe by-product formation. By-product formation of primary-amine specific ligations is due to unwanted reactions at numerous nucleophilic functional groups such as secondary amino, alcoholic and phenolic hydroxyl, carboxyl, etc present in both the ligating probes and the targeted multifunctional molecules/biological entities.
Several primary amine-specific ligation methodologies have been introduced in the past. These ligation processes have severe shortcomings regarding to the achieved substitution degree and selectivities.
The above mentioned primary amine ligation techniques often suffer from low degree of substitution or low degree of chemoselectivity due to the use of very reactive ligating probes such as mixed anhydrides. In several cases, the use of activating agents is also necessary complicating work-up procedures and lowering product purities (mixed anhydride method, reductive amination).
Furthermore, in some ligation methodologies toxic or hard to remove condensation by-products could form causing serious problems in the derivatisation of sensitive biological entities (2-iminomethoxymethylthio ligation, acyl azide ligation, squaric acid ligation). In most of the cases, the developed methodologies use linker systems containing artificial and/or toxic residues (coupling with aryl-isothiocyanates, squaric acid ligation) limiting the scope of ligations by the introduction of unnecessary linking moieties.
Thus, there is a demand for the development of new ligation methodologies suitable for conjugations of carbohydrates to proteins in view of the limitations of present technologies. Novel methodologies have to fulfill the following criteria:                The ligation reaction should preferably work in aqueous solutions, preferably in water        A direct linkage between the conjugated moieties should preferably be established, thereby eliminating the use of artificial linkers.        Natural and non-toxic linker moieties can be accepted.        Coupling reagents should be avoided during the ligation reaction.        Condensation by-product formation should be eliminated.        The ligation chemistry should be capable of working in a wide pH range.        The reactivity of ligating probes should support both chemoselectivity and site-selectivity while rapid conjugations could be achieved.        
EP 441192 A2 discloses retroisosteric dipeptides and their use as rennin inhibitors.
WO 88/02756 A2 discloses sugar derivatives of a biologically active peptide with prolonged duration of action.